(M u ltidrug resistance, M DR )
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- Σωτήρης Φωτόπουλος
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1 V o l. 26 N o CH EM ICAL JOU RNAL O F CH IN ESE UN IV ERS IT IES , 1, 2 (1., ; 2., ) V CR Bel27402 M DR,.,, V CR. Bel27402, V CR, V CR G2, P2170, M DR 1gmRNA, Topo gαmrna. ; ; ; ; R 735 A (2005) (M u ltidrug resistance, M DR ).,,. M DR,,. (P2 ) ; ;, P53 Bcl22; DNA [1 3 ]. M DR,,. T su ruo [4 ],, (Calcium channel b locker, CCB ),., CCB [5 ]. Bel27402,, H ew lett 1100 ; Cou lter Ep ics XL ; M odel 550 (B IO 2RAD ) ; 9700 PCR ; Kodak (EDA S). Bel27402 ( ) ; (BCDC), (V er2 apam il, V R P, ) ; (V incristine, V CR., ) ; P2gp JSB 21 ( ) ; AM V (GibcogBRL ) ; T ripure (GibcogBRL ) ; T aq ( ) ; ( ) M T T 0125%, 10% R PM I gml, 96, : : ( : ) ( : ). : (1962 ),,,,. E2m ail: gaixiaodong@ yahoo. com. cn
2 N o. 8 : 200 ΛL,, 6 : (1) BCDC. 5 ΛL BCDC 300, 320, 340, 350, 360, 380, 390, ΛggmL ; (2) BCDC+ V CR. BCDC 300, 320, 340, 350, 360, 380, 390, ΛggmL, 5 ΛL V CR ( 0102 ΛggmL ) ; (3) V R P+ V CR. V R P 5 ΛL ( 5 ΛggmL ), 5 ΛL V CR ( 0102 ΛggmL ) ; (4), ; (5) V CR. 5 ΛL V CR ( 0102 ΛggmL ) ; (6),. 37 5% CO 2 4 d, 50 ΛL M T T (1 m ggml ), 4 h,, 150 ΛL DM SO, 10 m in, OD 570 nm. : (% ) = OD OD 570 OD OD 570 = [ 6 ], CD I= A B g(a B ) CD I, A B, A B, CD I< 1, Bel27402 V CR [7 ] Bel ml, gml, 24 h, 3 : 110 ΛggmL ) ; (1) Bel V CR ( (2) Bel V CR ( 110 ΛggmL ) + V R P ( 5 Λgg ml ) ; (3) Bel V CR ( 110 ΛggmL ) + BCDC ( 390 ΛggmL ). 24 h, 900 rgm in 10 m in, 019% N ac l 3, 15 ml g, 900 rgm in 7 m in,, 114 ml DDW, , 900 rgm in 30 m in,, 4,. Zo rbax2od S, 0102 mo lgl K 2H PO 4 (ph = 616) 2 ( 20 80), 110 ml g m in, 50, 267 nm Bel gml, 80% 90%, : (1)Bel27402 ; ( 2)Bel V CR ( 0102 ΛggmL ) + V R P ( 5 Λggm l) ; (3) Bel BCDC ( 390 ΛggmL ) ; (4) Bel BCDC ( 390 ΛggmL ) + V CR ( 0102 ΛggmL ). 3,, 900 rgm in 5 m in,, gml, PBS 2, 5 ml 70%,, 4 24 h, rgm in 5 m in,, PBS 2, PBS 1 ml, RN ase, 50 ΛggmL, 37 1 h, (P I), 50 ΛggmL, 4, 30 m in, 200, P2gp S2P P2 (P2gp ) R T 2PCR M DR Bel % R PM I1640, 37 5% CO 2, gml. Bel27402 ; Bel ΛggmL BCDB, 24, 48, h M DR. T rizo l RNA, R T 2PCR, cdna PCR : M DR 1, M R P, GST Π, Topog Α Β2actin 1. : 94 3 m in, s, s, 72 1 m in, m in 30, 115%., M DR 1gΒ2actin, M R PgΒ2actin, GST ΠgΒ2actin Topog gβ2actin, M DR 1, M R P, GST Π Topog ΑmRNA. 1447
3 V o l. 26 Gene M DR1 Table 1 The sequence of pr imers and the length of multiplication products(bp) M ultip lication M ultip lication Sequence of p rim ers Gene Sequence of p rim ers p roduct (bp) p roduct (bp) 5 CCGCT GT TCGT T TCCT T TA GGT GST Π 5 CCGT GGTCTA T T TCCCA GT TCG TCT TCT T T GCTCCTCCA T T GCG3 5 CA GCAA GTCCA GCA GGT T GTA G3 Topog Α 5 GGGT T TA CGA T GAA GA T GT T GGC3 114 Β2A ctin 5 AA GA GA GGCA TCCT GA CCCT M RP 5 GGGTCCCT T T GT T T GT T GTCCG 3 5 A T GCTCA CT T TCT GGCT GGTA G A CCT T GGAA CTCTCT T TCGGCT Bel TA CA T GGCT GGGGT GT T GAA ΛggmL,,, P > 0105; 400 ΛggmL,, P < ΛggmL Bel27402 V CR,, P < 0101, Bel27402, 390 ΛggmL ( 2)., CD I 0189, 390 ΛggmL, 718, V R P A dded arug Cell Contro l V CR V RP+ V CR Table 2 D ose-dependen t exper imen t of BCDC reversal on Bel-7402 Cell L ine b lg(λg ml - 1 ) BCDC c b BCDC+ V CR d d d a. T he date are the m ean values SD, n= 6; b. P < 0101 as compared w ith the contro l cell; c. P < 0105, as compared w ith the con2 tro l cell; d. P < 0101 as compared w ith the cells treated by V CR Bel-7402 VCR, V CR, V CR, V CR m in,., ngg10 ΛL. 390 ΛggmL ( ) nggm 2 ( ) nggm 2, P < 0101, ( ) nggm 2. V CR V CR Bel-7402, (390 ΛggmL ),, V CR G % 37143% (P < 0101), V R P+ V CR G2 (42177% ), ( 3). Bel27402 M DR V CR, V CR,, G2 [8 ]. 10%,, Bel27402 V CR. Group Table 3 The effect of BCDC treatmen t on Cell Cycle a Cell Cycle (% ) G0G1 S G2M Bel Bel V RP+ V CR b Bel ΛggmL BCDC Bel ΛggmL DCDC+ V CR b a. T he cell cycle w as analyzed after 3 days of expo sure to the compounds as described in the experim ental m ethods. A ll data w ere the m ean value SD in trip licate. b. P < 0101 as compared w ith contro l cell group. 1448
![N o. 8 : 2. 4 P-gp P2gp [ 1 (A ) ],, Bel27402 P2gp [ 1 (B ) ]. M DR M DR 1 P2170. P2170,, [9, 10 ]. P2170,., F ig. 1 Expression of P-gp in Bel-7402 cells (A ) Bel27402 cells.](/docs-images/92/108934289/images/4-0.jpg "S2P m ethod 400; (B) Bel27402 cells after BCDC treatm ent fo r 3 d. S2P m ethod 400. 2. 5 4. M DR 1, GST Π Topog Α Bel27402, M R P 30. M DR 1 mrna, ; Topog Α, ; GST Π ( 2).")
4 N o. 8 : 2. 4 P-gp P2gp [ 1 (A ) ],, Bel27402 P2gp [ 1 (B ) ]. M DR M DR 1 P2170. P2170,, [9, 10 ]. P2170,., F ig. 1 Expression of P-gp in Bel-7402 cells (A ) Bel27402 cells. S2P m ethod 400; (B) Bel27402 cells after BCDC treatm ent fo r 3 d. S2P m ethod M DR 1, GST Π Topog Α Bel27402, M R P 30. M DR 1 mrna, ; Topog Α, ; GST Π ( 2). Table 4 The expression changes of mrna related to MD R in Bel-7402 cell treated with DCDC 3 Group T im egd DR 1 GST Π Topog Α Bel Bel BCDC 390 ΛggmL a a a a M ean SD, n= 3; a. P < 0101 as compared w ith contro l cell group at sam e tim e. M DR 1 P2gp,. M R P ABC, M R P [11 ]. GST, GST Π. [12 ], F ig. 2 The level of MD R1, GSTΠ and Topog Α gene transcr ipt in Bel-7402 and Bel-7402 cells treated by 390 ΛggmL BCDC (lane 1, 2, 3, 4, 5, 6 respectively) DNA DNA,. Topog,, DNA 2Topog. Topog, [3 ].., Bel27402 M R P, M DR 1gmRNA P2gp, Topog ΑmRNA, 1449
5 V o l. 26 Topog Α. 3, Bel27402 M DR, Bel27402 M DR 1gmRNA, P2, Topog Α mrna, Bel V CR V CR, V CR G2M.,,. [ 1 ] Endico tt J. A., L ing V.. A nn. Rev. B iochem. [J ], 1989, 58: 137_ 171 [ 2 ] T etu B., B risson J., P lante V. et a l.. M od. Patho l. [J ], 1998, 11 (9) : 823_ 830 [ 3 ] Pomm ier Y., L eteurte F., Fesen M. R. et a l.. Cancer Invesṫ [J ], 1994, 12: [ 4 ] T suruo T., L ida H., T sukago sh i S. et a l.. Cancer Res. [J ], 1981, 41: 1967_ 1972 [ 5 ] GAO X ian2jun ( ). Journal of Integrated T raditional Ch inese and W estern M edicine ( ) [J ], 1990, 10 (7) : 447_ 448 [ 6 ] SU J ian ( ), ZH EN Yong2Su ( ), Q I Chang2Q ing ( ) et al.. A cta Pharm aceutica Sinica ( ) [J ], 1994, 29: 656_ 661 [ 7 ] L I D an2rong ( ), TU W en2sheng ( ), L I L i( ) et al.. Ch inese Journal of Ch rom atography ( ) [J ], 1998, 16 (1) : 50_ 51 [ 8 ] L in H. L., L iu T. Y., W u C. W. et a l.. B r. J. Cancer[J ], 1999, 8 (13) : 416_ 422 [ 9 ] Scam bia G., Ranelletti F. O., Benedetti Panici P. et a l.. Cancer Chemo theṙ Pharmoco l. [J ], 1994, 34: 459_ 464 [ 10 ] L in H. L., L iu T. Y., L uiw. Y. et a l.. Cancer[J ], 1999, 85 (9) : 1937_ 1942 [ 11 ] Jedlitschky G., Kepp ler D.. V itam Ho rm [J ], 2002, 64: 153_ 184 [ 12 ] T ew K. D.. Cancer Res. [J ], 1994, 54: 4313_ 4320 Effect of Bupleurun Ch inese DC (BCDC) on the MD R Reversal of Hepatocellular Carc inoma and Related M echan ism GA I X iao2dong 13, ZEN G Chang2Q ian 1, HON G M in 2 (1. L aboratory of L if e S cience and B iolog ical T echnology, B eihua U niversity, J ilin , Ch ina; 2. M ed icine P rotein and P roteom ics laboratory, J ilin U niversity, Chang chun , Ch ina) Abstract T he reversal effect and m echan ism s of BCDC w ere evaluated by u sing Bel27402 cell line w ith innate resistance in th is study. T he iden tification of new and less tox ic reversal agen t, BCDC, w ou ld open a new perspectives in cancer treatm en ṫ T he resu lts show that BCDC has an effect on M DR reversal of Bel27402 cell line and a synergetic effect w ith V CR. T he reversal m echan ism s w ere due to supp ress M DR 1 tran scrip tion and P2gp exp ression, as a resu lt, to leave TopogΑmRNA level, to increase in tracellu lar V CR concen tration of Bo l27402 cell, to enhance cell G2M phase arrest induced by V CR. Keywords H epatocellu lar carcinom a; M u ltidrug resistance (M DR ) ; M DR reversal; V incristine (V CR ) ; Bup leu run Ch inese DC (Ed. : D, I) 1450
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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.
LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.
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Supplementary Table 1. Primers used for RT-qPCR analysis of striatal and nigral tissue.
Supplementary Table 1. Primers used for RT-qPCR analysis of striatal and nigral tissue. Gene Forward Primer (5-3 ) Reverse Primer (5-3 ) Dopaminergic Markers TH CTG GCC ATT GAT GTA CTG GA ACA CAC ATG GGA
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